Skip to main content
PMC home page
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1988 Nov 1;107(5):1777–1783. doi: 10.1083/jcb.107.5.1777

The reorganization of microfilaments, centrosomes, and microtubules during in vitro small wound reendothelialization

PMCID: PMC2115346  PMID: 3182937

Abstract

The repair of small endothelial wounds is an important process by which endothelial cells maintain endothelial integrity. An in vitro wound model system was used in which precise wounds were made in a confluent endothelial monolayer. The repair process was observed by time-lapse cinemicrophotography. Using fluorescence and immunofluorescence microscopy, the cellular morphological events were correlated with the localization and distribution of actin microfilament bundles and vinculin plaques, and centrosomes and their associated microtubules. Single to four-cell wounds underwent closure by cell spreading while wounds seven to nine cells in size closed by initially spreading which was then followed at approximately 1 h after wounding by cell migration. These two processes showed different cytoskeletal patterns. Cell spreading occurred independent of centrosome location. However, centrosome redistribution to the front of the cell occurred as the cells began to elongate and migrate. While the peripheral actin microfilament bundles (i.e., the dense peripheral band) remained intact during cell spreading, they broke down during migration and were associated with a reduction in peripheral vinculin plaque staining. Thus, the major events characterizing the closure of endothelial wounds were precise in nature, followed a specific sequence, and were associated with specific cytoskeletal patterns which most likely were important in maintaining directionality of migration and reducing the adhesion of the cells to their neighbors within the monolayer.

Full Text

The Full Text of this article is available as a PDF (2.8 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Albrecht-Buehler G., Bushnell A. The orientation of centrioles in migrating 3T3 cells. Exp Cell Res. 1979 Apr;120(1):111–118. doi: 10.1016/0014-4827(79)90542-1. [DOI] [PubMed] [Google Scholar]
  2. Albrecht-Buehler G. Phagokinetic tracks of 3T3 cells: parallels between the orientation of track segments and of cellular structures which contain actin or tubulin. Cell. 1977 Oct;12(2):333–339. doi: 10.1016/0092-8674(77)90109-x. [DOI] [PubMed] [Google Scholar]
  3. Anderson D. C., Wible L. J., Hughes B. J., Smith C. W., Brinkley B. R. Cytoplasmic microtubules in polymorphonuclear leukocytes: effects of chemotactic stimulation and colchicine. Cell. 1982 Dec;31(3 Pt 2):719–729. doi: 10.1016/0092-8674(82)90326-9. [DOI] [PubMed] [Google Scholar]
  4. Bergmann J. E., Kupfer A., Singer S. J. Membrane insertion at the leading edge of motile fibroblasts. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1367–1371. doi: 10.1073/pnas.80.5.1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Bergmann J. E., Tokuyasu K. T., Singer S. J. Passage of an integral membrane protein, the vesicular stomatitis virus glycoprotein, through the Golgi apparatus en route to the plasma membrane. Proc Natl Acad Sci U S A. 1981 Mar;78(3):1746–1750. doi: 10.1073/pnas.78.3.1746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Connolly J. A., Kalnins V. I., Cleveland D. W., Kirschner M. W. Immunoflourescent staining of cytoplasmic and spindle microtubules in mouse fibroblasts with antibody to tau protein. Proc Natl Acad Sci U S A. 1977 Jun;74(6):2437–2440. doi: 10.1073/pnas.74.6.2437. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. De Brabander M. A model for the microtubule organizing activity of the centrosomes and kinetochores in mammalian cells. Cell Biol Int Rep. 1982 Oct;6(10):901–915. doi: 10.1016/0309-1651(82)90001-7. [DOI] [PubMed] [Google Scholar]
  8. De Brabander M. Microtubules, central elements of cellular organization. Endeavour. 1982;6(3):124–134. doi: 10.1016/0160-9327(82)90045-x. [DOI] [PubMed] [Google Scholar]
  9. Dylewski D. P., Keenan T. W. Centrioles in the mammary epithelium of the rat. J Cell Sci. 1984 Dec;72:185–193. doi: 10.1242/jcs.72.1.185. [DOI] [PubMed] [Google Scholar]
  10. Erickson C. A., Trinkaus J. P. Microvilli and blebs as sources of reserve surface membrane during cell spreading. Exp Cell Res. 1976 May;99(2):375–384. doi: 10.1016/0014-4827(76)90595-4. [DOI] [PubMed] [Google Scholar]
  11. Euteneuer U., Schliwa M. Persistent, directional motility of cells and cytoplasmic fragments in the absence of microtubules. Nature. 1984 Jul 5;310(5972):58–61. doi: 10.1038/310058a0. [DOI] [PubMed] [Google Scholar]
  12. Follett E. A., Goldman R. D. The occurrence of microvilli during spreading and growth of BHK21-C13 fibroblasts. Exp Cell Res. 1970 Jan;59(1):124–136. doi: 10.1016/0014-4827(70)90631-2. [DOI] [PubMed] [Google Scholar]
  13. Gabbiani G., Gabbiani F., Lombardi D., Schwartz S. M. Organization of actin cytoskeleton in normal and regenerating arterial endothelial cells. Proc Natl Acad Sci U S A. 1983 Apr;80(8):2361–2364. doi: 10.1073/pnas.80.8.2361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Geiger B., Rosen D., Berke G. Spatial relationships of microtubule-organizing centers and the contact area of cytotoxic T lymphocytes and target cells. J Cell Biol. 1982 Oct;95(1):137–143. doi: 10.1083/jcb.95.1.137. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Gordon S. R., Essner E., Rothstein H. In situ demonstration of actin in normal and injured ocular tissues using 7-nitrobenz-2-oxa-1,3-diazole phallacidin. Cell Motil. 1982;2(4):343–354. doi: 10.1002/cm.970020404. [DOI] [PubMed] [Google Scholar]
  16. Gotlieb A. I., May L. M., Subrahmanyan L., Kalnins V. I. Distribution of microtubule organizing centers in migrating sheets of endothelial cells. J Cell Biol. 1981 Nov;91(2 Pt 1):589–594. doi: 10.1083/jcb.91.2.589. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Gotlieb A. I., Spector W. Migration into an in vitro experimental wound: a comparison of porcine aortic endothelial and smooth muscle cells and the effect of culture irradiation. Am J Pathol. 1981 May;103(2):271–282. [PMC free article] [PubMed] [Google Scholar]
  18. Gotlieb A. I., Spector W., Wong M. K., Lacey C. In vitro reendothelialization. Microfilament bundle reorganization in migrating porcine endothelial cells. Arteriosclerosis. 1984 Mar-Apr;4(2):91–96. doi: 10.1161/01.atv.4.2.91. [DOI] [PubMed] [Google Scholar]
  19. Gotlieb A. I., Subrahmanyan L., Kalnins V. I. Microtubule-organizing centers and cell migration: effect of inhibition of migration and microtubule disruption in endothelial cells. J Cell Biol. 1983 May;96(5):1266–1272. doi: 10.1083/jcb.96.5.1266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Herman B., Pledger W. J. Platelet-derived growth factor-induced alterations in vinculin and actin distribution in BALB/c-3T3 cells. J Cell Biol. 1985 Apr;100(4):1031–1040. doi: 10.1083/jcb.100.4.1031. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kreis T. E., Birchmeier W. Stress fiber sarcomeres of fibroblasts are contractile. Cell. 1980 Nov;22(2 Pt 2):555–561. doi: 10.1016/0092-8674(80)90365-7. [DOI] [PubMed] [Google Scholar]
  22. Kupfer A., Dennert G., Singer S. J. Polarization of the Golgi apparatus and the microtubule-organizing center within cloned natural killer cells bound to their targets. Proc Natl Acad Sci U S A. 1983 Dec;80(23):7224–7228. doi: 10.1073/pnas.80.23.7224. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Kupfer A., Louvard D., Singer S. J. Polarization of the Golgi apparatus and the microtubule-organizing center in cultured fibroblasts at the edge of an experimental wound. Proc Natl Acad Sci U S A. 1982 Apr;79(8):2603–2607. doi: 10.1073/pnas.79.8.2603. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Malech H. L., Root R. K., Gallin J. I. Structural analysis of human neutrophil migration. Centriole, microtubule, and microfilament orientation and function during chemotaxis. J Cell Biol. 1977 Dec;75(3):666–693. doi: 10.1083/jcb.75.3.666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Mascardo R. N., Sherline P. Glyburide modulates insulin-mediated locomotor response of confluent cell cultures to wounding. Diabetes. 1987 Dec;36(12):1476–1482. doi: 10.2337/diab.36.12.1476. [DOI] [PubMed] [Google Scholar]
  26. Mascardo R. N., Sherline P. Insulin and multiplication-stimulating activity induce a very rapid centrosomal orientation response to wounding in endothelial cell monolayers. Diabetes. 1984 Nov;33(11):1099–1105. doi: 10.2337/diab.33.11.1099. [DOI] [PubMed] [Google Scholar]
  27. Nemere I., Kupfer A., Singer S. J. Reorientation of the Golgi apparatus and the microtubule-organizing center inside macrophages subjected to a chemotactic gradient. Cell Motil. 1985;5(1):17–29. doi: 10.1002/cm.970050103. [DOI] [PubMed] [Google Scholar]
  28. Pollard T. D., Selden S. C., Maupin P. Interaction of actin filaments with microtubules. J Cell Biol. 1984 Jul;99(1 Pt 2):33s–37s. doi: 10.1083/jcb.99.1.33s. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Reidy M. A., Schwartz S. M. Endothelial injury and regeneration. IV. Endotoxin: a nondenuding injury to aortic endothelium. Lab Invest. 1983 Jan;48(1):25–34. [PubMed] [Google Scholar]
  30. Reidy M. A., Schwartz S. M. Recent advances in molecular pathology. Arterial endothelium--assessment of in vivo injury. Exp Mol Pathol. 1984 Dec;41(3):419–434. doi: 10.1016/0014-4800(84)90031-5. [DOI] [PubMed] [Google Scholar]
  31. Rinnerthaler G., Geiger B., Small J. V. Contact formation during fibroblast locomotion: involvement of membrane ruffles and microtubules. J Cell Biol. 1988 Mar;106(3):747–760. doi: 10.1083/jcb.106.3.747. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Rogalski A. A., Bergmann J. E., Singer S. J. Effect of microtubule assembly status on the intracellular processing and surface expression of an integral protein of the plasma membrane. J Cell Biol. 1984 Sep;99(3):1101–1109. doi: 10.1083/jcb.99.3.1101. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Rogalski A. A., Singer S. J. Associations of elements of the Golgi apparatus with microtubules. J Cell Biol. 1984 Sep;99(3):1092–1100. doi: 10.1083/jcb.99.3.1092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Rogers K. A., Boden P., Kalnins V. I., Gotlieb A. I. The distribution of centrosomes in endothelial cells of non-wounded and wounded aortic organ cultures. Cell Tissue Res. 1986;243(2):223–227. doi: 10.1007/BF00251035. [DOI] [PubMed] [Google Scholar]
  35. Rogers K. A., McKee N. H., Kalnins V. I. Preferential orientation of centrioles toward the heart in endothelial cells of major blood vessels is reestablished after reversal of a segment. Proc Natl Acad Sci U S A. 1985 May;82(10):3272–3276. doi: 10.1073/pnas.82.10.3272. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Schliwa M., Pryzwansky K. B., van Blerkom J. Implications of cytoskeletal interactions for cellular architecture and behavior. Philos Trans R Soc Lond B Biol Sci. 1982 Nov 4;299(1095):199–205. doi: 10.1098/rstb.1982.0126. [DOI] [PubMed] [Google Scholar]
  37. Shasby D. M., Shasby S. S., Sullivan J. M., Peach M. J. Role of endothelial cell cytoskeleton in control of endothelial permeability. Circ Res. 1982 Nov;51(5):657–661. doi: 10.1161/01.res.51.5.657. [DOI] [PubMed] [Google Scholar]
  38. Singer I. I. Association of fibronectin and vinculin with focal contacts and stress fibers in stationary hamster fibroblasts. J Cell Biol. 1982 Feb;92(2):398–408. doi: 10.1083/jcb.92.2.398. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Soni S. L., Gotlieb A. I., Kalnins V. I. In vitro spreading of porcine aortic endothelial and smooth muscle cells. Scan Electron Microsc. 1980;(3):263–270. [PubMed] [Google Scholar]
  40. Swanson J. A., Taylor D. L. Local and spatially coordinated movements in Dictyostelium discoideum amoebae during chemotaxis. Cell. 1982 Feb;28(2):225–232. doi: 10.1016/0092-8674(82)90340-3. [DOI] [PubMed] [Google Scholar]
  41. White G. E., Gimbrone M. A., Jr, Fujiwara K. Factors influencing the expression of stress fibers in vascular endothelial cells in situ. J Cell Biol. 1983 Aug;97(2):416–424. doi: 10.1083/jcb.97.2.416. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Wong A. J., Pollard T. D., Herman I. M. Actin filament stress fibers in vascular endothelial cells in vivo. Science. 1983 Feb 18;219(4586):867–869. doi: 10.1126/science.6681677. [DOI] [PubMed] [Google Scholar]
  43. Wong M. K., Gotlieb A. I. Endothelial cell monolayer integrity. I. Characterization of dense peripheral band of microfilaments. Arteriosclerosis. 1986 Mar-Apr;6(2):212–219. doi: 10.1161/01.atv.6.2.212. [DOI] [PubMed] [Google Scholar]
  44. Wong M. K., Gotlieb A. I. In vitro reendothelialization of a single-cell wound. Role of microfilament bundles in rapid lamellipodia-mediated wound closure. Lab Invest. 1984 Jul;51(1):75–81. [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES